The release of cytochrome c from mitochondria is often recognized as the commitment step of apoptosis. The mitochondrial apoptosis-induced channel (MAC) forms early in apoptosis in hematopoietic cells, concurrent with Bax translocation into mitochondria. Our data show MAC could provide a pathway for cytochrome c to exit mitochondria and over-expression of anti-apoptotic Bcl-2 suppresses this channel activity. There is a gap in knowledge defining the relationship between BCL-2 family proteins and MAC, e.g. are Bak and Bax components of MAC? The hypothesis of this proposal is that MAC provides a pathway for cytochrome c, and perhaps other proteins in the mitochondrial intermembrane space, to exit the mitochondria during apoptosis and MAC is a critical target for regulation of the mitochondrial apoptotic response by members of the BCL-2 family of proteins. This proposal is aimed at testing this hypothesis using a combination of electrophysiological, molecular biological and microscopic techniques. Aim 1 is to systematically characterize the channel properties of MAC and its permeability to cytochrome c, thereby testing whether MAC can permit the release of cytochrome c from mitochondria early in apoptosis. We plan to determine if cytochrome c transports through MAC using electrophysiology and ELISA. Aim 2 is to determine if pro-apoptotic Bax and Bak are integral, structural components of MAC, and will be tested, in part, by assessing MAC activity in mitochondria of cells that are knockouts for Bax and/or Bak, which will also determine if there is more than one kind of MAC activity. Aim 3 is to determine the mechanism(s) by which pro-apoptotic BH3-only and anti-apoptotic Bcl-2/Bcl-xL proteins regulate MAC formation. We hypothesize that pro- and anti-apoptotic proteins act by antagonistically regulating formation of MAC. We plan to determine if BH3-only proteins promote the formation of MAC and if anti-apoptotic BCL2 family members can inhibit the formation or activity of MAC. One approach will be to assess the development of MAC activity while patch-clamping mitochondria in the presence of Bcl-2/Bcl-xL and/or representative BH3-only proteins. These experiments will provide fundamental insights into the nature of mitochondrial involvement in apoptosis and the regulation by BCL-2 family proteins. These experiments may identify novel therapeutic targets, e.g., MAC, to modulate apoptosis in cancer and other pathologies, e.g., myocardial infarction.